Cellular protooncogenes encode proteins that are thought to regulate normal cellular proliferation and differentiation. Alterations in their structure or amplification of their expression lead to abnormal cellular growth and have been associated with carcinogenesis (Bishop J. M., Science 235:305-311 [1987]); (Rhims J. S., Cancer Detection and Prevention 11:139-149 [1988]); (Nowell P. C., Cancer Res. 46:2203-2207 [1986]); (Nicolson G. L., Cancer Res. 47:1473-1487 [1987]). Protooncogenes were first identified by either of two approaches. First, molecular characterization of the genomes of transforming retroviruses showed that the genes responsible for the transforming ability of the virus in many cases were altered versions of genes found in the genomes of normal cells. The normal version is the protooncogene, which is altered by mutation to give rise to the oncogene. An example of such a gene pair is represented by the EGF receptor and the v-erb-B gene product. The virally encoded v-erb-B gene product has suffered truncation and other alterations that render it constitutively active and endow it with the ability to induce cellular transformation (Yarden et al, Ann. Rev. Biochem. 57:443-478, 1988).
The second method for detecting cellular transforming genes that behave in a dominant fashion involves transfection of cellular DNA from tumor cells of various species into nontransformed target cells of a heterologous species. Most often this was done by transfection of human, avian, or rat DNAs into the murine NIH 3T3 cell line (Bishop J. M., Science 235:305-311 [1987]); (Rhims J. S., Cancer Detection and Prevention 11:139-149 [1988]); (Nowell P. C., Cancer. Res. 46:2203-2207 [1986]); (Nicolson G. L., Cancer. Res. 47:1473-1487 [1987]); (Yarden et al., Ann. Rev. Biochem. 57:443-478 [1988]). Following several cycles of genomic DNA isolation and retransfection, the human or other species DNA was molecularly cloned from the murine background and subsequently characterized. In some cases, the same genes were isolated following transfection and cloning as those identified by the direct characterization of transforming viruses. In other cases, novel oncogenes were identified. An example of a novel oncogene identified by this transfection assay is the neu oncogene. It was discovered by Weinberg and colleagues in a transfection experiment in which the initial DNA was derived from a carcinogen-induced rat neuroblastoma (Padhy et al., Cell 28:865-871 [1982]); (Schechter et al., Nature 312:513-516 [1984]). Characterization of the rat neu oncogene revealed that it had the structure of a growth factor receptor tyrosine kinase, had homology to the EGF receptor, and differed from its normal counterpart, the neu protooncogene, by an activating mutation in its transmembrane domain (Bargmann et al., Cell 45:649-657 [1986]). The human counterpart to neu is the HER2 protooncogene, also designated c-erb- B2 (Coussens et al., Science 230:1137-1139 [1985]); U.S. Ser. No. 07/143,912).
The association of the HER2 protooncogene with cancer was established by yet a third approach, that is, its association with human breast cancer. The HER2 protooncogene was first discovered in cDNA libraries by virtue of its homology with the EGF receptor, with which it shares structural similarities throughout (Yarden et al., Ann. Rev. Biochem. 57:443-478 [1988]). When radioactive probes derived from the cDNA sequence encoding p185.sup.HER2 were used to screen DNA samples from breast cancer patients, amplification of the HER2 protooncogene was observed in about 30% of the patient samples (Slamon et al., Science 235:177-182 [1987]). Further studies have confirmed this original observation and extended it to suggest an important correlation between HER2 protooncogene amplification and/or overexpression and worsened prognosis in ovarian cancer and non-small cell lung cancer (Slamon et al., Science 244:707-712 [1989]); (Wright et al., Cancer Res 49:2087-2090, 1989); (Paik et al., J. Clin. Oncology 8:103-112 [1990]); (Berchuck et al., Cancer Res. 50:4087-4091, 1990); (Kern et al., Cancer Res. 50:5184-5191, 1990).
The association of HER2 amplification/overexpression with aggressive malignancy, as described above, implies that it may have an important role in progression of human cancer; however, many tumor-related cell surface antigens have been described in the past, few of which appear to have a direct role in the genesis or progression of disease (Schlom et al. Cancer Res. 50:820-827, 1990); (Szala et al., Proc. Natl. Acad. Sci. 98:3542-3546).
Among the protooncogenes are those that encode cellular growth factors which act through endoplasmic kinase phosphorylation of cytoplasmic protein. The HER1 gene (or erb-B1) encodes the epidermal growth factor (EGF) receptor. The .beta.-chain of platelet-derived growth factor is encoded by the c-sis gene. The granulocyte-macrophage colony stimulating factor is encoded by the c-fms gene. The neu protooncogene has been identified in ethylnitrosourea-induced rat neuroblastomas. The HER2 gene encodes the 1,255 amino acid tyrosine kinase receptor-like glycoprotein p185.sup.HER 2 that has homology to the human epidermal growth factor receptor.
The known receptor tyrosine kinases all have the same general structural motif: an extracellular domain that binds ligand, and an intracellular tyrosine kinase domain that is necessary for signal transduction and transformation. These two domains are connected by a single stretch of approximately 20 mostly hydrophobic amino acids, called the transmembrane spanning sequence. This transmembrane spanning sequence is thought to play a role in transferring the signal generated by ligand binding from the outside of the cell to the inside. Consistent with this general structure, the human p185.sup.HER2 glycoprotein, which is located on the cell surface, may be divided into three principal portions: an extracellular domain, or ECD (also known as XCD); a transmembrane spanning sequence; and a cytoplasmic, intracellular tyrosine kinase domain. While it is presumed that the extracellular domain is a ligand receptor, the p185.sup.HER2 ligand has not yet been positively identified.
No specific ligand binding to p185.sup.HER2 has been identified, although Lupu et al., (Science 249:1552-1555, 1989) describe an inhibitory 30 kDa glycoprotein secreted from human breast cancer cells which is alleged to be a putative ligand for p185.sup.HER2 . Lupu et al., Science, 249:1552-1555 (1990); Proceedings of the American Assoc. for Cancer Research, Vol 32, Abs 297, March 1991) reported the purification of a 30 kD factor from MDA-MB-231 cells and a 75 kD factor from SK-BR-3 cells that stimulates p185.sup.HER2 . The 75 kD factor reportedly induced phosphorylation of p185.sup.HER2 and modulated cell proliferation and colony formation of SK-BR-3 cells overexpressing the p185.sup.HER2 receptor. The 30 kD factor competes with muMab 4D5 for binding to p185.sup.HER2 , its growth effect on SK-BR-3 cells was dependent on 30 kD concentration (stimulatory at low concentrations and inhibitory at higher concentrations). Furthermore, it stimulated the growth of MDA-MB-468 cells (EGF-R positive, p185.sup.HER2 negative), it stimulated phosphosylation of the EGF receptor and it could be obtained from SK-BR-3 cells. In the rat neu system, Yarden et al., (Biochemistry, 30:3543-3550, 1991) describe a 35 kDa glycoprotein candidate ligand for the neu encoded receptor secreted by ras transformed fibroblasts. Dobashi et al., Proc. Natl. Acad. Sci. USA, 88:8582-8586 (1991); Biochem. Biophys. Res. Commun.; 179:1536-1542 (1991) described a neu protein-specific activating factor (NAF) which is secreted by human T-cell line ATL-2 and which has a molecular weight in the range of 8-24 kD. A 25 kD ligand from activated macrophages was also described (Tarakhovsky, et al., J. Cancer Res., 2188-2196 (1991).
Methods for the in vivo assay of tumors using HER2 specific monoclonal antibodies and methods of treating tumor cells using HER2 specific monoclonal antibodies are described in U.S. Ser. No. 07/143,912.
There is a current and continuing need in the art to identify the actual ligand or ligands that activate p185.sup.HER2 , and to identify their biological role(s), including their roles in cell-growth and differentiation, cell-transformation and the creation of malignant neoplasms.
Accordingly, it is an object of this invention to identify and purify one or more novel p185.sup.HER2 ligand polypeptide(s) that bind and stimulate p185.sup.HER2.
It is another object to provide nucleic acid encoding novel p185.sup.HER2 binding ligand polypeptides and to use this nucleic acid to produce a p185.sup.HER2 binding ligand polypeptide in recombinant cell culture for therapeutic or diagnostic use, and for the production of therapeutic antagonists for use in certain metabolic disorders including, but not necessarily restricted to the killing, inhibition and/or diagnostic imaging of tumors and tumorigenic cells.
It is a further object to provide derivatives and modified forms of novel glycoprotein ligands, including amino acid sequence variants, fusion polypeptides combining a p185.sup.HER2 binding ligand and a heterologous protein and covalent derivatives of a p185.sup.HER2 binding ligand.
It is an additional object to prepare immunogens for raising antibodies against p185.sup.HER2 binding ligands, as well as to obtain antibodies capable of binding to such ligands, and antibodies which bind a p185.sup.HER2 binding ligand and prevent the ligand from activating p185.sup.HER2. It is a further object to prepare immunogens comprising a p185.sup.HER2 binding ligand fused with an immunogenic heterologous polypeptide.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.